A typical reaction assembling three components using the hygromycin B phosphotransferase gene (hph) as a selective marker. The hph gene was amplified with primers, HPH-F (5′-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3′ and HPH-R 5′-AACCCGCGGTCGGCATCTACTCTATTC-3′) from pBIG2RHPH2-GFP-GUS. The upstream (∼1,100 bp) and downstream (∼1,400 bp) flanking sequences of VmEP1 were amplified using primer pairs VmEP1-1F/2R and VmEP1-3F/4R, respectively. Then the deletion cassette was generated by double-joint PCR as described (Yu et al., 2004 (link)), using the primer pair VmEP1-CF/CR. For generating deletion mutants, the VmEP1 gene-replacement construct was transformed into protoplasts of V. mali as previously described (Gao et al., 2011 ). Putative deletion mutants were verified by PCR using four primer pairs (VmEP1-5F/6R, VmEP1-7F/H855R, VmEP1-H856F/8R, and H850/H852) to detect target gene (∼850 bp), upstream (∼1,100 bp), and downstream (∼1,400 bp) region, and the hph gene (∼750 bp), respectively. Subsequently, deletion mutants were confirmed by Southern blot hybridization using the DIG DNA Labeling and Detection Kit II (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Primers used for gene deletion are listed in Supplementary Table S2 .
Dig dna labeling and detection kit 2
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The DIG DNA Labeling and Detection Kit II is a laboratory product designed for the labeling and detection of DNA. It provides the necessary reagents and tools to enable the incorporation of digoxigenin (DIG) into DNA samples, allowing for their subsequent detection and visualization.
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5 protocols using dig dna labeling and detection kit 2
1
Deletion of VmEP1 Gene in V. mali
2
Single Gene Deletion in Valsa mali
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A reaction with three components using the Neo gene as a selective marker was performed for single gene deletion. The Neo gene was amplified with primers Neo-F and Neo-R from PFL2. The upstream and downstream flanking sequences of VmPxE1 were amplified using primer pairs 1F/2R, 3F/4R, respectively. Then, deletion cassette for homologous recombination was generated by double-joint PCR as described previously (Yu et al., 2004 (link)). The primer pair CF/CR was used for nest-PCR and produced the gene-replacement construct. Protoplasts of V. mali were prepared and then the gene-replacement construct was transformed into the protoplasts as previously described (Gao et al., 2011 (link)). Each putative single gene deletion mutant was verified by PCR using four primer pairs (5F/6R, 7F/NeoR, NeoF/8R, and NeoF/NeoR) to detect the target gene, upstream-neo fusion segment, neo-downstream fusion segment, and the neo gene, respectively. Southern blot hybridization using the DIG DNA Labeling and Detection Kit II (Roche, Mannheim, Germany) was performed to confirm deletion mutants. All primers used for gene deletion are given in Supplementary Table S1 .
3
Targeted Gene Knockout and Complementation in Valsa mali
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VmLaeA and the core enzyme genes of cluster PKS7, PKS11, NRPS14, PKS16, PKS23, PKS31, NRPS/PKS33, and PKS39 were selected to construct deletion mutants. The gene knockout cassettes were assembled by double-joint PCR with upstream and downstream flanking sequences of the target gene and neo gene as selective marker (Yu et al., 2004 (link); Supplementary Figure 8 ). The gene knockout cassettes were then transformed into protoplasts of V. mali mediated by polyethylene glycol (Gao et al., 2011 ). Putative deletion mutants filtered by selection with geneticin were confirmed by PCR and further confirmed by Southern blot hybridization using the DIG DNA Labeling and Detection Kit II (Roche, Mannheim, Germany). Gene complementation was conducted by cloning the target genes into plasmid PDL2 by the yeast gap repair approach and then transforming the recombined plasmids into the respective target gene deletion mutant (Bruno et al., 2004 (link); Zhou et al., 2012 (link)). The complemented mutants were finally confirmed by PCR. All primers used in gene deletion and complementation are listed in Supplementary Table 11 .
4
Targeted Deletion and Complementation of CfEC92
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The upstream and downstream flanking sequences of CfEC92 were amplified. The resulting PCR products were fused with the hygromycin phosphotransferase (hph) fragments by double‐jointed PCR (Figure S1a). The joined upstream and downstream products were then transformed into protoplasts of C. fructicola. Transformants resistant to hygromycin were examined by PCR. Further deletion mutants were confirmed by Southern blot hybridization using the DIG DNA Labeling and Detection Kit II (Roche) according to the manufacturer's instructions. To complement the CfEC92‐deficient strain, the recombinant pHZ‐100‐CfEC92 was introduced into protoplasts of ΔCfEC92‐27 mutant strain. Complementation strains were identified from G418‐resistant transformants using PCR.
5
Northern Blot Analysis of GhUXS3 mRNA
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The expression of GhUXS3 mRNA in transgenic Arabidopsis was analyzed by northern blot analysis. Total RNA was extracted from positive T 3 transgenic lines and one 'Columbia-0' wild-type (WT) control plant, using TRIZOL reagent (TransGen, China). The extracted RNA was combined with 8 µL formaldehyde and 5 µL 10X MOPS (morpholinopropanesulfonic acid, 200 mM MOPS, 50 mM sodium acetate, 20 mM EDTA, pH 7.0) and heated at 65°C for 10 min. Thereafter, the samples were cooled on ice for 5 min, 6 µL 10X loading buffer was added to them and they were separated on agarose gels containing 10X MOPS. The electrophoresed RNA was transferred by capillary blotting onto Hybond N + nylon membranes (Amersham Pharmacia, USA). Probes for the RNA gel blot were PCR-amplified from the pBI121:GhUXS3 chimeric vector with the primers 5'-CCCCACCCACGAGGAGCAT-3' and 5'-TCCATCCACAACCCGAGACATACT-3'. The digoxigenin-labeling of the probe, its denaturation, and northern blot analysis were performed with a DIG DNA Labeling and Detection Kit II (Roche, Germany) according to the manufacturer instructions. The membranes were hybridized overnight at 42°C and washed twice, for 20 min each, in 2X SSC and 0.1% SDS followed by two washes with 0.5X SSC and 0.1% SDS at 65°C; the membranes were subsequently exposed to X-ray film.
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